Difference between revisions of "Part:BBa K203105"
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===Usage and Biology===
 
===Usage and Biology===
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Low-density lipoprotein receptor (LDL receptor) is a cell surface protein which is responsible for the cholesterol supply of the cell. The receptor recognizes the protein B100 which is part of the LDL particles. Binding of the B100 protein to the LDL receptor leads to endocytosis via clathrin coated pits. The vesicle fuses with an endosome, the resulting shift in the pH value leads to the detachment of the LDL. The receptor can be transported back to the plasma membrane (receptor recycling). Through this process the cell takes up the cholesterol which is associated with LDL. LDL accumulation in the blood is responsible for Atherosclerosis and many cardiovascular diseases. The LDL gene is regulated by the intracellular level of cholesterol. A low level of cholesterol leads to the activation whereas a high level results in a decrease of transcription. See reference at [http://2009.igem.org/Team:Heidelberg/Eucaryopedia#Proteins Eucaryopedia].
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[[Image:HD09_SREBP.png|thumb|left|300px|'''SREBP induction.''' Upon Sterol depletion, the interaction of SCAP and Insig in the ER membrane is inhibited, resulting in cleavage of SREBP, and migration to the nucleus. [http://en.wikipedia.org/wiki/File:WikF1.png Image is public domain.]]]
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Sterol regulatory element-binding protein (SREBP) is a transcription factor involved in the regulation of sterol metabolism. In cells with high concentration of cholestrol SREBP is present in an inactive form anchored to the endoplasmatic reticulum or the nuclear envelop. If the cholesterol concentration decreases SREBP is cleaved by the proteases site-1 protease and site-2 protease resulting in a release of the aminoterminal domain of SREBP. Two additional proteins (Scap and Insig) are needed to regulate this process in a way that the cleavage occurs exclusively during lack of sterol. The aminoterminal domain of SREBP is translocated into the nucleus and binds to the DNA consensus sequence TCACNCCAC. The binding causes an up regulation of the genes needed for cholesterol synthesis. For references, see [http://2009.igem.org/Team:Heidelberg/Eukaryopedia#SREBP Eukaryopedia].
  
  
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<partinfo>BBa_K203105 parameters</partinfo>
 
<partinfo>BBa_K203105 parameters</partinfo>
  
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The natural LDL receptor promoter was induced by Lipoprotein Deficient Serum (more details: see [http://2009.igem.org/wiki/index.php?title=Team:Heidelberg/Notebook_promoters_cells&action=edit&redlink=1 Cell Culture Notebook]) in [http://2009.igem.org/Team:Heidelberg/Eucaryopedia#HeLa HeLa cells]. Promoter activtiy was then roughly characterized analogous to [http://2009.igem.org/Team:Heidelberg/Measurement REU] by TECAN (automated plate fluorescence reader). For more natural promoters look at [[Part:BBa_K203106]] and [[Part:BBa_K203107]].  
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The natural LDL receptor promoter was integrated into [[Part:BBa_K203100]] and was induced by Lipoprotein Deficient Serum (LDS) in [http://2009.igem.org/Team:Heidelberg/Eucaryopedia#HeLa HeLa cells]. The 0,5% LDS DMEM (1% Glucosamin, 1% Pen/Strep, 1% non-essential aminoacids) medium was put on cells after transfection for two days. After that, 1% hydroxypropyl-β-cyclodextrin (HPCD) medium was put on for 3 hours and then, 0,5% LDS DMEM (1% Glucosamin, 1% Pen/Strep, 1% non-essential aminoacids) medium was used for incubation for 3 hours. Promoter activtiy was then roughly characterized analogous to [http://2009.igem.org/Team:Heidelberg/Measurement REU] by TECAN (automated plate fluorescence reader).
  
 
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|[[Image:HD09_LDL_receptor.png|thumb|none|350px|'''BBa_K203105 is a natural LDL receptor promoter induced approximately 25% by Lipoprotein Deficient Serum;'''. The GFP flourescence of the LDL receptor promoter was measured by TECAN once (à three replicates). Background fluorescence was substracted, and fluorescence levels are plotted relative to [[Part:BBa_K203112]].]]
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|[[Image:HD09_LDL_receptor.png|thumb|none|350px|'''BBa_K203105 is a natural LDL receptor promoter induced approximately 25% by Lipoprotein Deficient Serum;'''. The LDL receptor promoter is coupled to the fluorecent protein GFP. This GFP fluorescence was measured by TECAN once (à three replicates). Background fluorescence was substracted, and fluorescence levels are plotted relative to [[Part:BBa_K203112]].The standard deviation is represented by the error bars. Inactiving condition is full medium with extra LDL and Cholesterol added]]
 
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Latest revision as of 18:49, 21 October 2009

LDL receptor promoter (sterol regulated)

This part is a natural LDL receptor promoter responsive to SREBP and when placed upstream of other genes could induce their transcription in response to lipid addition to the medium.

Usage and Biology

SREBP induction. Upon Sterol depletion, the interaction of SCAP and Insig in the ER membrane is inhibited, resulting in cleavage of SREBP, and migration to the nucleus. [http://en.wikipedia.org/wiki/File:WikF1.png Image is public domain.]

Sterol regulatory element-binding protein (SREBP) is a transcription factor involved in the regulation of sterol metabolism. In cells with high concentration of cholestrol SREBP is present in an inactive form anchored to the endoplasmatic reticulum or the nuclear envelop. If the cholesterol concentration decreases SREBP is cleaved by the proteases site-1 protease and site-2 protease resulting in a release of the aminoterminal domain of SREBP. Two additional proteins (Scap and Insig) are needed to regulate this process in a way that the cleavage occurs exclusively during lack of sterol. The aminoterminal domain of SREBP is translocated into the nucleus and binds to the DNA consensus sequence TCACNCCAC. The binding causes an up regulation of the genes needed for cholesterol synthesis. For references, see [http://2009.igem.org/Team:Heidelberg/Eukaryopedia#SREBP Eukaryopedia].


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 139
    Illegal SapI site found at 128

Functional Parameters

n/aLDL receptor promoter (sterol regulated)

The natural LDL receptor promoter was integrated into Part:BBa_K203100 and was induced by Lipoprotein Deficient Serum (LDS) in [http://2009.igem.org/Team:Heidelberg/Eucaryopedia#HeLa HeLa cells]. The 0,5% LDS DMEM (1% Glucosamin, 1% Pen/Strep, 1% non-essential aminoacids) medium was put on cells after transfection for two days. After that, 1% hydroxypropyl-β-cyclodextrin (HPCD) medium was put on for 3 hours and then, 0,5% LDS DMEM (1% Glucosamin, 1% Pen/Strep, 1% non-essential aminoacids) medium was used for incubation for 3 hours. Promoter activtiy was then roughly characterized analogous to [http://2009.igem.org/Team:Heidelberg/Measurement REU] by TECAN (automated plate fluorescence reader).

BBa_K203105 is a natural LDL receptor promoter induced approximately 25% by Lipoprotein Deficient Serum;. The LDL receptor promoter is coupled to the fluorecent protein GFP. This GFP fluorescence was measured by TECAN once (à three replicates). Background fluorescence was substracted, and fluorescence levels are plotted relative to Part:BBa_K203112.The standard deviation is represented by the error bars. Inactiving condition is full medium with extra LDL and Cholesterol added